Detergent compositions containing substantially pure EG III cellulase

ABSTRACT

Disclosed are detergent compositions containing a cleaning effective amount of a surfactant or a mixture of surfactants and from about 0.01 to about 5 weight percent of substantially pure EG III cellulase. Preferably, the detergent composition contains no more than about 5 weight percent of CBH I type components based on the total weight of cellulase proteins. When employed in aqueous wash media, the detergent compositions impart color retention/restoration properties as well as improved softening and feel properties to cotton-containing fabrics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/079,546, filed Jun. 22, 1993, now U.S. Pat. No. 5,419,778, which is adivisional of U.S. patent application Ser. No. 07/707,647 filed on May30, 1991, now U.S. Pat. No. 5,290,474, which is a continuation-in-partof U.S. patent application Ser. No. 07/688,640 filed Mar. 13, 1991, nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 07/593,919, filed Oct. 5, 1990, now abandoned, the disclosureof these latter two applications are incorporated herein in theirentirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to detergent compositions containingsubstantially pure EG III cellulose from Trichoderma spp. as well as tomethods for employing such compositions. In particular, the detergentcompositions of the present invention comprise a cleaning effectiveamount of one or more surfactants and substantially pure EG IIIcellulase.

2. State of the Art

Cellulases are known in the art as enzymes that hydrolyze cellulose(β-1,4-glucan linkages) thereby resulting in the formation of glucose,cellobiose, cellooligosaccharides, and the like. While cellulases areproduced (expressed) in fungi, bacteria and the like, cellulase producedby certain fungi and, in particular by the fungal class Trichoderma Sp.(especially Trichoderma longibrachiatum), have been given the mostattention because a complete cellulase system capable of degradingcrystalline forms of cellulose is readily produced in large quantitiesvia fermentation procedures.

In regard to the above, Schulein., "Methods in Enzymology", 160, 25,pages 234 et seq. (1988), disclose that complete fungal cellulasesystems comprise several different enzyme classifications includingthose identified as exo-cellobiohydrolases (EC 3.2.1.91) ("CBH"),endoglucanases (EC 3.2.1.4) ("EG"), and β-glucosidases (EC 3.2.1.21)("BG"). The fungal cellulose classifications of CBH, EG and BG can befurther expanded to include multiple components within eachclassification. For example, multiple CBHs and EGS have been isolatedfrom a variety of fungal sources.

The complete cellulase system comprising CBH, EG and BG components isrequired to efficiently convert crystalline cellulose to glucose.Isolated components are far less effective, if at all, in hydrolyzingcrystalline cellulose. Moreover, a synergistic relationship is observedbetween the cellulase components particularly if they are of differentclassification.

On the other hand, cellulases and components thereof, used eithersingularly or in combination, are also known in the art to be useful indetergent compositions. For example, endoglucanase components of fungalcellulases have been used for the purposes of enhancing the cleaningability of detergent compositions, for use as a softening agent, and foruse in improving the feel of cotton fabrics, and the like. However,there is a problem with using the EG I and EG II components derived fromTrichoderma spp. and especially Trichoderma longibrachiatum in detergentcompositions. Specifically, such components have their maximal activityat acidic pHs whereas most laundry detergent compositions are formulatedfor use at neutral or alkaline (pH>7 to about 10) conditions. Whiles itis disclosed in U.S. Ser. No. 07/668,640 that the use of one or moreacidic endoglucanase components of Trichoderma longibrachiatum indetergent compositions will provide improvements in softening, colorretention/restoration and feel to cotton-containing fabrics even whentreated under alkaline conditions, the present invention is directed tothe discovery that the EG III component of Trichoderma spp. provides forsuperior and unexpected advantages in detergent compositions as comparedto the EG I and EG II components of Trichoderma longibrachiatum.

Specifically, this component has been found to possess significantenzymatic activity under alkaline conditions.

SUMMARY OF THE INVENTION

The present invention is directed to the use of substantially pure EGIII cellulase in detergent compositions to attain improvements insoftening, color retention/restoration and feel. Specifically, becauseof the surprisingly enhanced activity of EG III cellulose under alkalineconditions, detergent compositions containing substantially pure EG IIIcellulase are particularly suited for use in laundry conditions where aneutral or alkaline detergent wash medium is employed.

Accordingly, in one of its composition aspects, the present invention isdirected to a detergent composition comprising a cleaning effectiveamount of a surfactant or a mixture of surfactants and from about 0.01to about 5 weight percent, and preferably from about 0.05 to about 2weight percent, of a substantially pure EG III cellulase.

In one of its method aspects, the present invention is directed to amethod for enhancing the softness of a cotton-containing fabric whichmethod comprises washing the fabric in a wash medium derived from adetergent composition comprising a cleaning effective amount of asurfactant or a mixture of surfactants and from about 0.01 to about 5,and preferably from about 0.05 to about 2, weight percent ofsubstantially pure EG III cellulase.

In another of its method aspects, the present invention is directed to amethod for retaining/restoring the color of a cotton-containing fabricwhich method comprises washing the fabric one or more times in a washmedium derived from a detergent composition comprising a cleaningeffective amount of a surfactant or a mixture of surfactants and fromabout 0.01 to about 5, and preferably from about 0.05 to about 2, weightpercent of substantially pure EG III cellulase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the RBB-CMC activity profile over a pH range at 40°C. for an EG enriched fungal cellulase composition derived from a strainof Trichoderma longibrachiatum transformed so as to be incapable ofexpressing CBH I and CBH II; as well as the activity profile of anenriched EG III cellulase composition derived from Trichodermalongibrachiatum over a pH range at 40° C.

FIG. 2 is an isoelectric focusing gel which, in Lane 1, displays theproteins expressed by a wild type Trichoderma longibrachiatum; in Lane 2displays the proteins expressed by a strain of Trichodermalongibrachiatum transformed so as to be incapable of expressing EG I andEG II components; and in Lane 3 displays the proteins found insubstantially pure EG III cellulase. The left hand margin of this figureis marked so as to identify the bands attributable to CBH I, CBH II, EGI, EG II and EG III.

FIG. 3 illustrates the fiber removal properties and hence the colorrestoration properties of EG III at various pH's.

FIG. 4 is an outline of the construction of pΔCBHIpyr4.

FIG. 5 illustrates deletion of the Trichoderma longibrachiatum cbh1 geneby integration of the larger EcoRI fragment from pΔCBHIpyr4 at the cbh1locus on one of the Trichoderma longibrachiatum chromosomes.

FIG. 6 is an autoradiograph of DNA from a Trichoderma longibrachiatumstrain GC69 transformed with EcoRI digested pΔCBHIpyr4 after Southernblot analysis using a ³² P labelled pΔCBHIpyr4 as the probe.

FIG. 7 is an autoradiograph of DNA from a Trichoderma longibrachiatumstrain GC69 transformed with EcoRI digested pΔCBHIpyr4 after Southernblot analysis using a ³² P labelled pIntCBHI as the probe.

FIG. 8 is an isoelectric focusing gel displaying the proteins secretedby the wild type and by transformed strains of Trichodermalongibrachiatum. Specifically, in FIG. 8, Lane A of the isoelectricfocusing gel employs partially purified CBH I from Trichodermalongibrachiatum; Lane B employs protein from a wild type Trichodermalongibrachiatum; Lane C employs protein from a Trichodermalongibrachiatum strain with the cbh1 gene deleted; and Lane D employsprotein from a Trichoderma longibrachiatum strain with the cbh1 and cbh2genes deleted.

In FIG. 8, the right hand side of the figure is marked to indicate thelocation of the single proteins found in one or more of the secretedproteins. Specifically, BG refers to β-glucosidase; E1 refers toendoglucanase I; E2 refers to endoglucanase II; E3 refers toendoglucanase III; C1 refers to exo-cellobiohydrolase I; and C2 refersto exo-cellobiohydrolase II.

FIG. 9A is a representation of the Trichoderma longibrachiatum cbh2locus cloned as a 4.1 kB EcoRI fragment of genomic DNA and FIG. 9B is arepresentation of the cbh2 gene deletion vector, pPΔCBHII.

FIG. 10 is an autoradiograph of DNA from a Trichoderma longibrachiatumstrain P37PΔCBHI transformed with EcoRI digested pPΔCBHII after Southernblot analysis using a ³² P labelled pPΔCBHII as the probe.

FIG. 11 is a diagram of the plasmid pEGI pyr4.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention generally relates to detergentcompositions containing substantially pure EG III cellulose as well asfor methods employing such detergent compositions. When used in aqueouswash media at alkaline pH's, such compositions are particularlyeffective in imparting improvements in softening, colorretention/restoration, and feel to the treated cotton-containing fabric.

However, prior to discussing this invention in detail, the followingterms will first be defined.

The term "EG III cellulase" refers to the endoglucanase componentderived from Trichoderma spp. characterized by a pH optimum of about 5.5to 6.0, an isoelectric point (pI) of from about 7.2 to 8.0, and amolecular weight of about 23 to 28 Kdaltons. Preferably, EG IIIcellulase is derived from either Trichoderma longibrachiatum or fromTrichoderma viride. EG III cellulase derived from Trichodermalongibrachiatum has a pH optimum of about 5.5 to 6.0, an isoelectricpoint (pI) of about 7.4 and a molecular weight of about 25 to 28Kdaltons. EG III cellulase derived from Trichoderma viride has a pHoptimum of about 5.5, an isoelectric point (pI) of about 7.7 and amolecular weight of about 23.5 Kdaltons.

"Substantially pure EG III cellulase" refers to a composition ofcellulase proteins containing at least 40 weight percent, preferably atleast 70 weight percent and most preferably at least 90 weight percentof EG III based on the total weight of cellulase proteins.

EG III cellulase can be purified from any strain of Trichoderma spp.which produces EG III under suitable fermentation conditions. While theparticular source of EG III is not critical, preferred sources areTrichoderma longibrachiatum and Trichoderma viride. A particularlypreferred source of EG III from Trichoderma longibrachiatum is Cytolase123 cellulase which is commercially available from GenencorInternational, Inc., 180 Kimball Way, South San Francisco, Calif. 94080.Because of its high pI, EG III is found in a region of an isoelectricfocusing gel where high pI xylanases and other high pI componentsexpressed by Trichoderma spp. are generally found. In fact, it has beenhypothesized that the band identified as EG III in FIG. 2 was adegradation product of either EG I or II. However, gel isoelectricfocusing of EG I and EG II deleted cellulase (prepared in the manner ofU.S. Ser. Nos. 07/593,919 and 07/668,640) demonstrated that this bandwas not attributable to a degradation product of either EG I or II. (SeeFIG. 2).

It is noted that EG II has been previously referred to by thenomenclature "EG III" by some authors but current nomenclature uses theterm "EG II". In any event, the EG II protein is substantially differentfrom the EG III protein in its molecular weight, pI, and pH optimum asevidenced by Table I of Example 2 presented below.

Procedures suitable for obtaining substantially pure EG III cellulasefrom a complete cellulase system derived from Trichoderma spp. ("wholecellulose") include those recited in the examples set forth hereinbelow. These examples demonstrate that substantially pure EG IIIcellulase is readily obtained by subjecting whole cellulase topurification procedures including repeated fractionation steps utilizingdifferent fractionation materials (columns). Additionally, thefractionation steps can be preceded by an extraction step usingpolyethylene glycol 8000 so as to provide for EG III cellulase fraction(about 20-50% pure EG III) which, if necessary, can be used insubsequent fractionation steps to provide for substantially pure EG IIIcellulase.

Additionally, it is contemplated that substantially pure EG IIIcellulase can be prepared by genetically modifying microorganisms so asto produce substantially pure EG III cellulase. For example,substantially pure EG III prepared by fractionation methods set forth inthe examples below can be employed to determine the amino acid sequenceof parts or all of the protein using known sequencing methods. Once theamino acid sequence of parts of the EG III cellulase is known, thisinformation can be used to prepare synthetic DNA probes in order toclone the gene responsible for encoding this information. Once the EGIII encoding gene is cloned, it could be manipulated by recognizedtechniques and ultimately inserted into various Trichoderma spp. strainsor into other microorganisms. See, for example, U.S. Ser. No.07/593,919, filed Oct. 5, 1990 and U.S. Ser. No. 07/668,640, filed Mar.13, 1991, both of which disclose methods for genetically engineeringTrichoderma longibrachiatum so that the modified microorganism isincapable of expressing one or more of the cellulase genes and, in fact,may overproduce another cellulase gene. Using the methods described inthese applications, Trichoderma longibrachiatum could be geneticallymanipulated so as to produce EG III with or without other cellulaseproteins. Moreover, the methods of these applications create Trichodermalongibrachiatum strains which do not produce any heterologous proteins.The disclosures of both U.S. Ser. No. 07/593,919, filed Oct. 5, 1990 andU.S. Ser. No. 07/668,640, filed Mar. 13, 1991, are incorporated hereinby reference in their entirety.

Additionally, it would be possible to express the EG III-encoding genein other microorganisms, including, but not limited to, yeast speciessuch as Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha,Kluyveromyces lactis, Yarrowia lipolytica, Schanniomyces occidentalis,etc. See, for example, PCT application Publication No. WO 85/04672. Inorder to obtain expression in these alternative, non-Trichoderma hosts,it may be necessary to functionally combine the EG III-coding DNAsequence with promoter and terminator sequences obtained from a genefrom that particular host. It may also be necessary to substitute theDNA sequence encoding a secretion signal sequence from the alternativehost for the DNA sequence encoding the EG III secretion signal sequence.Production and secretion of EG III in other organisms could enable EGIII to be obtained in substantially pure form.

"Cellulase proteins" refer to any and all exo-cellobiohydrolase (CBH)proteins, endoglucanase (EG) proteins and β-glucosidase (BG) proteinsexpressed by a wild type Trichoderma spp. or a transformed strain ofTrichoderma spp. Accordingly, cellulase proteins do not include otherproteins expressed by Trichoderma spp. including xylanases, proteases,amylases, etc.

"Exo-cellobiohydrolase ("CBH") components" refer to the CBH I and/or CBHII components of Trichoderma longibrachiatum and "CBH type components"refer to those fungal cellulase components which exhibit detergentactivity properties similar to that of the CBH I and/or CBH IIcomponents of Trichoderma longibrachiatum. In this regard, when used inthe absence of the EG components of Trichoderma longibrachiatum, the CBHI and CBH II components of Trichoderma longibrachiatum alone do notimpart significant color retention/restoration and improved feel to theso-treated cotton-containing fabrics. Additionally, when used incombination with such EG components, the CBH I component of Trichodermalongibrachiatum can impart enhanced strength loss and incrementalcleaning benefits to cotton-containing fabrics.

Accordingly, CBH I type components and CBH II type components refer tothose fungal cellulase components which exhibit detergent activityproperties similar to CBH I and CBH II components of Trichodermalongibrachiatum, respectively. As noted above, for CBH I typecomponents, this includes the properties of enhancing strength loss ofcotton-containing fabrics and/or imparting an incremental cleaningbenefit when used in the presence of the EG components of Trichodermalongibrachiatum. In a preferred embodiment, the CBH I components alsoimpart an incremental softening benefit when used in the presence ofsuch EG components.

Such CBH type components may exclude components traditionally classifiedas exo-cellobiohydrolases using activity tests such as those used tocharacterize CBH I and CBH II from Trichoderma longibrachiatum. Forexample, using such traditional classification tests, such components(a) are competitively inhibited by cellobiose (K_(i) approximately 1mM); (b) hydrolyze phosphoric acid swollen cellulose and to a lesserdegree highly crystalline cellulose; and (c) are unable to hydrolyze toany significant degree substituted celluloses, such ascarboxymethylcellulose, etc. In contrast, it is believed that some CBHcomponents which are characterized as exo-cellobiohydrolase componentsby such activity tests, do not possess functional properties similar tothe CBH components of Trichoderma longibrachiatum. Accordingly, it isbelieved to be more accurate for the purposes herein not to define suchexo-cellobiohydrolases as CBH type components because these componentsdo not possess similar functional properties in detergent compositionsas possessed by the CBH components of Trichoderma longibrachiatum.

"β-Glucosidase (BG) components" refer to those components of cellulasewhich exhibit BG activity; that is, to say that such components will actfrom the non-reducing end of cellobiose and other solublecellooligosaccharides ("cellobiose") and give glucose as the soleproduct. BG components do not adsorb onto or react with cellulosepolymers. Furthermore, such BG components are competitively inhibited byglucose (K_(i) approximately 1 mM). While in a strict sense, BGcomponents are not literally cellulases because they cannot degradecellulose, such BG components are included within the definition of thecellulase system because these enzymes facilitate the overalldegradation of cellulose by further degrading the inhibitory cellulosedegradation products (particularly cellobiose) produced by the combinedaction of CBH components and EG components. Without the presence of BGcomponents, moderate or little hydrolysis of crystalline cellulose willoccur. BG components are often characterized on aryl substrates such asp-nitrophenol B-D-glucoside (PNPG) and thus are often calledaryl-glucosidases. It should be noted that not-all aryl-glucosidases areBG components, in that some do not hydrolyze cellobiose.

In some cases, it may be desirable to incorporate BG components into thedetergent compositions of this invention (e.g., so as to increase theoverall hydrolysis of cellulose if the level of cellobiose becomesrestrictive of such overall hydrolysis in the absence of BG components).In such cases, purified BG components can be added to the detergentcomposition. The amount of BG component added depends upon the amount ofcellobiose produced in the detergent wash which can be readilydetermined by the skilled artisan. However, when employed, the weightpercent of BG component relative to total weight of substantially pureEG III cellulase is preferably from about 0.4 to about 10 weightpercent, and more preferably, from about 1 to about 5 weight percent.

Fungal cellulases can contain more than one BG component and aregenerally separated via ion exchange chromatography and the like. Inorder to facilitate separation, it may be desirable to employ acellulase composition having increased amounts of BG. Methods toincrease the amount of BG components in the cellulase composition aredisclosed in U.S. Ser. No. 07/625,140, filed Dec. 10, 1990, and entitled"SACCHARIFICATION OF CELLULOSE BY CLONING AND AMPLIFICATION OF THEβ-GLUCOSIDASE GENE OF TRICHODERM REESEI", which application isincorporated herein by reference.

The term "cotton-containing fabric" refers to sewn or unsewn fabricsmade of pure cotton or cotton blends including cotton woven fabrics,cotton knits, cotton denims, and the like. When cotton blends areemployed, the amount of cotton in the fabric should be at least about 40percent by weight cotton; preferably, more than about 60 percent byweight cotton; and most preferably, more than about 75 percent by weightcotton. When employed as blends, the companion material employed in thefabric can include one or more non-cotton fibers including syntheticfibers such as polyamide fibers (for example, nylon 6 and nylon 66),acrylic fibers (for example, polyacrylonitrile fibers), polyester fibers(for example, polyethylene terephthalate), polyvinyl alcohol fibers (forexample, Vinylon), polyvinyl chloride fibers, polyvinylidene chloridefibers, polyurethane fibers, polyurea fibers and aramid fibers. It iscontemplated that regenerated cellulose, such as rayon, could be used asa substitute for cotton in cotton-containing fabrics.

The term "surface active agent or surfactant" refers to anionic,non-ionic and ampholytic surfactants well known for their use indetergent compositions.

The term "wash medium" refers to an aqueous wash solution prepared byadding a requisite amount of a detergent (surfactant) composition towater. The wash medium generally contains a cleaning effective amount ofthe detergent.

The wash medium is defined as an "acidic wash mediums" if the pH of themedium is from about 4 to less than about 7. The wash medium is definedas a "neutral wash medium" if the pH of the medium is about 7. The washmedium is defined as an "alkaline wash medium" if the pH of the mediumis from above 7 to about 10. Preferably, the alkaline wash medium willhave a pH of from above 7 to about 9 and, even more preferably, fromabove 7 to about 8.

The present invention is directed to the discovery that substantiallypure EG III cellulase can be used in detergent compositions to effectsoftening as well as effecting color retention/restoration and improvedfeel of cotton-containing fabrics regardless of whether suchcompositions are employed in an acidic, neutral or alkaline wash medium.However, because EG III possesses unexpectedly high activity underalkaline conditions, detergent compositions employing substantially pureEG III cellulase provide enhanced softening, color retention/restorationand improved feel as compared to detergent compositions employing otherendoglucanase components derived from Trichoderma longibrachiatum.

Although the presence of EG III cellulase effects colorretention/restoration, softening and improved feel, specified mixturesof EG III cellulase with other cellulase components can provide someincremental benefits. Specifically, while the use of EG III cellulasewill provide a cleaning and softening benefit, incremental cleaning andsoftening benefits are observed for cotton-containing fabrics washedwith a detergent composition containing EG III cellulase and CBH I typecellulose.

On the other hand, the presence of significant amounts of CBH I typecomponents in combination with EG III cellulase may result in enhancedstrength loss to cotton-containing fabrics compared to EG IIIcompositions which are either free of CBH I type components or containreduced amounts of CBH I type components. Accordingly, when thedetergent composition contains some CBH I type components, the amount ofCBH I type components is preferably no greater than about 10 weightpercent, more preferably no greater than about 5 weight percent, andeven more preferably less than about 2 weight percent, based on thetotal weight of cellulose proteins.

In regard to the above, the total amount of substantially pure EG IIIcellulase generally employed in the detergent compositions of thisinvention is an amount sufficient to impart color retention/restorationand softness to the cotton garments. Preferably, the substantially pureEG III cellulase composition is employed from about 0.01 weight percentto about 5 weight percent relative to the weight of the total detergentcomposition. More preferably, substantially pure EG III is employed fromabout 0.05 weight percent to about 2 weight percent relative to theweight of the total detergent composition. In general, the amount ofother cellulase proteins employed in the detergent composition is nomore than about 60 weight percent relative to the total weight ofcellulase proteins (including EG III cellulase), preferably no more thanabout 30 weight percent relative to the total weight of celluloseproteins, and more preferably no more than about 10 weight percentrelative to the total weight of cellulase proteins. Even morepreferably, the CBH type I components preferably do not exceed about 5weight percent of the total weight of cellulase proteins (i.e.,substantially pure EG III cellulose).

The specific concentration of substantially pure EG III cellulaseemployed in the detergent composition is selected so that upon dilutioninto a wash medium, the concentration of EG III cellulase willpreferably range from about 0.5 to about 500 ppm, and more preferablyfrom about 2 to about 100 ppm. The specific amount of substantially pureEG III cellulase employed in the detergent composition will depend onthe extent the detergent composition will be diluted upon addition towater to form a wash medium. These factors are readily ascertained bythe skilled artisan.

At lower cellulase concentrations (i.e., concentrations of EG IIIcellulase of less than about 5 ppm in the wash medium), softness, colorretention/restoration and improved feel achieved by use of the detergentcompositions of this invention is more evident over repeated washings.At higher concentrations (i.e., concentrations of EG III cellulase ofabout 5 ppm and above in the wash medium), the improvements can becomenoticeable in a single wash.

One of the important aspects of the present invention is that bytailoring the cellulase composition to contain substantially pure EG IIIcellulase, it is possible to achieve the desired effects of softening,color retention/restoration while using lower concentrations ofcellulase in the detergent composition. In turn, the use of lowerconcentrations of cellulase in the detergent compositions should lead toimproved consumer safety.

The substantially pure EG III cellulase described above can be added tothe detergent composition either in a liquid diluent, in granules, inemulsions, in gels, in pastes, or the like. Such forms are well known tothe skilled artisan. When a solid detergent composition is employed, thecellulase composition is preferably formulated as granules. Preferably,the granules can be formulated so as to contain a cellulase protectingagent. See, for instance, U.S. Ser. No. 07/642,669 filed Jan. 17, 1991as Attorney Docket No. 010055-073 and entitled "GRANULES CONTAINING BOTHAN ENZYME AND AN ENZYME PROTECTING AGENT AND DETERGENT COMPOSITIONSCONTAINING SUCH GRANULES" which application is incorporated herein byreference in its entirety. Likewise, the granule can be formulated so asto contain materials to reduce the rate of dissolution of the granuleinto the wash medium. Such materials and granules are disclosed in U.S.Ser. No. 07/642,596 filed on Jan. 17, 1991 as Attorney Docket No.GCS-171-US1 and entitled "GRANULAR COMPOSITIONS" which application isincorporated herein by reference in its entirety.

The detergent compositions of this invention employ a surface activeagent, i.e., surfactant, including anionic, non-ionic and ampholyticsurfactants well known for their use in detergent compositions.

Suitable anionic surfactants for use in the detergent composition ofthis invention include linear or branched alkylbenzenesulfonates; alkylor alkenyl ether sulfates having linear or branched alkyl groups oralkenyl groups; alkyl or alkenyl sulfates; olefinsulfonates;alkanesulfonates and the like. Suitable counter ions for anionicsurfactants include alkali metal ions such as sodium and potassium;alkaline earth metal ions such as calcium and magnesium; ammonium ion;and alkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3.

Ampholytic surfactants include quaternary ammonium salt sulfonates,betaine-type ampholytic surfactants, and the like. Such ampholyticsurfactants have both the positive and negative charged groups in thesame molecule, Nonionic surfactants generally comprise polyoxyalkyleneethers, as well as higher fatty acid alkanolamides or alkylene oxideadduct thereof, fatty acid glycerine monoesters, and the like.

Suitable surfactants for use in this invention are disclosed in BritishPatent Application No. 2 094 826 A, the disclosure of which isincorporated herein by reference.

Mixtures of such surfactants can also be used.

The surfactant or a mixture of surfactants is generally employed in thedetergent compositions of this invention in an amount from about 1weight percent to about 95 weight percent of the total detergentcomposition and preferably from about 5 weight percent to about 45weight percent of the total detergent composition. Upon dilution in thewash medium, the surfactant concentration is generally about 500 ppm ormore; and preferably, from about 1000 ppm to 15,000 ppm.

In addition to the cellulase composition and the surfactant(s), thedetergent compositions of this invention can optionally contain one ormore of the following components:

Hydrolases except cellulase

Such hydrolases include carboxylate ester hydrolase, thioesterhydrolase, phosphate monoester hydrolase, and phosphate diesterhydrolase which act on the ester bond; glycoside hydrolase which acts onglycosyl compounds; an enzyme that hydrolyzes N-glycosyl compounds;thioether hydrolase which acts on the ether bond; andα-amino-acyl-peptide hydrolase, peptidyl-amino acid hydrolase,acyl-amino acid hydrolase, dipeptide hydrolase, and peptidyl-peptidehydrolase which act on the peptide bond. Preferable among them arecarboxylate ester hydrolase, glycoside hydrolase, and peptidyl-peptidehydrolase. Suitable hydrolases include (1) proteases belonging topeptidyl-peptide hydrolase such as pepsin, pepsin B, rennin, trypsin,chymotrypsin A, chymotrypsin B, elastase, enterokinase, cathepsin C,papain, chymopapain, ficin, thrombin, fibrinolysin, renin, subtilisin,aspergillopeptidase A, collagenase, clostridiopeptidase B, kallikrein,gastrisin, cathepsin D., bromelin, keratinase, chymotrypsin C, pepsin C,aspergillopeptidase B, urokinase, carboxypeptidase A and B, andaminopeptidase; (2) glycoside hydrolases (cellulase which is anessential ingredient is excluded from this group) α-amylase, β-amylase,glucoamylase, invertase, lysozyme, pectinase, chitinase, and dextranase.Preferably among them are α-amylase and β-amylase. They function in acidto neutral systems, but one which is obtained from bacteria exhibitshigh activity in an alkaline system; (3) carboxylate ester hydrolaseincluding carboxyl esterase, lipase, pectin esterase, andchlorophyllase. Especially effective among them is lipase.

Trade names of commercial products and producers are as follows:"Alkalase", "Esperase", "Savinase", "AMG", "BAN", "Fungamill","Sweetzyme", "Thermamyl" (Novo Industry, Copenhagen, Denmark);"Maksatase", "High-alkaline protease", "Amylase THC", "Lipase" (GistBrocades, N.V., Delft, Holland); "Protease B-400", "Protease B-4000","Protease AP", "Protease AP 2100"(Schweizerische Ferment A.G., Basel,Switzerland); "CRD Protease" (Monsanto Company, St. Louis, Mo.);"Piocase" (Piopin Corporation, Monticello, Ill.); "Pronase P", "PronaseAS", "Pronase AF" (Kaken Chemical Co., Ltd., Japan); "Lapidase P-2000"(Lapidas, Secran, France); protease products (Tyler standard sieve, 100%pass 16 mesh and 100% on 150 mesh) (Clington Corn Products, Division ofStandard Brands Corp., New York); "Takamine", "Bromelain 1:10", "HTProtease 200", "Enzyme L-W" (obtained from fungi, not from bacteria)(Miles Chemical Company, Elkhart, Ind.); "Rhozyme P-11 Conc.","Pectinol", "Lipase B", "Rhozyme PF", "Rhozyme J-25" (Rohm & Haas,Genencor, South San Francisco, Calif.); "Ambrozyme 200" (Jack Wolf &Co., Ltd., Subsidiary of Nopco Chemical Company, Newark, N.J.); "ATP40", "ATP 120", "ATP 160" (Lapidas, Secran, France); "Oripase" (Nagase &Co., Ltd., Japan).

The hydrolase other than cellulase is incorporated into the detergentcomposition as much as required according to the purpose. It shouldpreferably be incorporated in an amount of 0.001 to 5 weight percent,and more preferably 0.02 to 3 weight percent, in terms of purified one.This enzyme should be used in the form of granules made of crude enzymealone or in combination with other components in the detergentcomposition. Granules of crude enzyme are used in such an amount thatthe purified enzyme is 0.001 to 50 weight percent in the granules. Thegranules are used in an amount of 0.002 to 20 and preferably 0.1 to 10weight percent. As with cellulases, these granules can be formulated soas to contain an enzyme protecting agent and a dissolution retardantmaterial.

Cationic surfactants and long-chain fatty acid salts

Such cationic surfactants and long-chain fatty acid salts includesaturated or unsaturated fatty acid salts, alkyl or alkenyl ethercarboxylic acid salts, α-sulfofatty acid salts or esters,amino acid-typesurfactants, phosphate ester surfactants, quaternary ammonium saltsincluding those having 3 to 4 alkyl substituents and up to 1 phenylsubstituted alkyl substituents. Suitable cationic surfactants andlong-chain fatty acid salts are disclosed in British Patent ApplicationNo. 2 094 826 A, the disclosure of which is incorporated herein byreference. The composition may contain from about 1 to about 20 weightpercent of such cationic surfactants and long-chain fatty acid salts.

Builders

A. Divalent sequestering agents

The composition may contain from about 0 to about 50 weight percent ofone or more builder components selected from the group consisting ofalkali metal salts and alkanolamine salts of the following compounds:phosphates, phosphonates, phosphonocarboxylates, salts of amino acids,aminopolyacetates high molecular electrolytes, non-dissociatingpolymers, salts of dicarboxylic acids, and aluminosilicate salts.Suitable divalent sequestering agents are disclosed in British PatentApplication No. 2 094 826 A, the disclosure of which is incorporatedherein by reference.

B. Alkalis or inorganic electrolytes

The composition may contain from about 1 to about 50 weight percent,preferably from about 5 to about 30 weight percent, based on thecomposition of one or more alkali metal salts of the following compoundsas the alkalis or inorganic electrolytes: silicates, carbonates andsulfates as well as organic alkalis such as triethanolamine,diethanolamine, monoethanolamine and triisopropanolamine.

Antiredeposition agents

The composition may contain from about 0.1 to about 5 weight percent ofone or more of the following compounds as antiredeposition agents:polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone andcarboxymethylcellulose.

Among them, a combination of carboxymethyl-cellulose or/and polyethyleneglycol with the cellulose composition of the present invention providesfor an especially useful dirt removing composition.

Bleaching agents

The use of the cellulase of the present invention in combination with ableaching agent such as sodium percarbonate, sodium perborate, sodiumsulfate/hydrogen peroxide adduct and sodium chloride/hydrogen peroxideadduct or/and a photo-sensitive bleaching dye such as zinc or aluminumsalt of sulfonated phthalocyanine further improves the detergingeffects.

Bluing agents and fluorescent dyes

Various bluing agents and fluorescent dyes may be incorporated in thecomposition, if necessary. Suitable bluing agents and fluorescent dyesare disclosed in British Patent Application No. 2 094 826 A, thedisclosure of which is incorporated herein by reference.

Caking inhibitors

The following caking inhibitors may be incorporated in the powderydetergent: p-toluenesulfonic acid salts, xylenesulfonic acid salts,acetic acid salts, sulfosuccinic acid salts, talc, finely pulverizedsilica, clay, calcium silicate (such as Micro-Cell of Johns ManvilleCo.), calcium carbonate and magnesium oxide.

Masking agents for factors inhibiting the cellulase activity

The cellulase compositions of this invention are deactivated in somecases in the presence of copper, zinc, chromium, mercury, lead,manganese or silver ions or their compounds. Various metal chelatingagents and metal-precipitating agents are effective against theseinhibitors. They include, for example, divalent metal ion sequesteringagents as listed in the above item with reference to optional additivesas well as magnesium silicate and magnesium sulfate.

Cellobiose, glucose and gluconolactone act sometimes as the inhibitors.It is preferred to avoid the co-presence of these saccharides with thecellulase as far as possible. In case the co-presence is unavoidable, itis necessary to avoid the direct contact of the saccharides with thecellulase by, for example, coating them.

Long-chain-fatty acid salts and cationic surfactants act as theinhibitors in some cases. However, the co-presence of these substanceswith the cellulase is allowable if the direct contact of them isprevented by some means such as tableting or coating.

The above-mentioned masking agents and methods may be employed, ifnecessary, in the present invention.

Cellulase-activators

The activators vary depending on variety of the cellulases. In thepresence of proteins, cobalt and its salts, magnesium and its salts, andcalcium and its salts, potassium and its salts, sodium and its salts ormonosaccharides such as mannose and xylose, the cellulases are activatedand their deterging powers are improved remarkably.

Antioxidants

The antioxidants include, for example, tert-butyl-hydroxytoluene,4,4'-butylidenebis(6-tert-butyl-3-methylphenol),2,2'-butylidenebis(6-tert-butyl-4-methylphenol), monostyrenated cresol,distyrenated cresol, monostyrenated phenol, distyrenated phenol and1,1-bis(4-hydroxy-phenyl)cyclohexane.

Solubilizers

The solubilizers include, for example, lower alcohols such as ethanol,benzenesulfonate salts, lower alkylbenzenesulfonate salts such asp-toluenesulfonate salts, glycols such as propylene glycol,acetylbenzene-sulfonate salts, acetamides, pyridinedicarboxylic acidamides, benzoate salts and urea.

The detergent composition of the present invention can be used in abroad pH range of from acidic to alkaline pH. In a preferred embodiment,the detergent composition of the present invention can be used inalkaline detergent wash media and more preferably, alkaline detergentwash media having a pH of from above 7 to no more than about 8.

Aside from the above ingredients, perfumes, buffers, preservatives, dyesand the like can be used, if desired, with the detergent compositions ofthis invention. Such components are conventionally employed in amountsheretofore used in the art.

When a detergent base used in the present invention is in the form of apowder, it may be one which is prepared by any known preparation methodsincluding a spray-drying method and a granulation method. The detergentbase obtained particularly by the spray-drying method and/orspray-drying granulation method are preferred. The detergent baseobtained by the spray-drying method is not restricted with respect topreparation conditions. The detergent base obtained by the spray-dryingmethod is hollow granules which are obtained by spraying an aqueousslurry of heat-resistant ingredients, such as surface active agents andbuilders, into a hot space. The granules have a size of from 50 to 2000micrometers. After the spray-drying, perfumes, enzymes, bleachingagents, inorganic alkaline builders may be added. With a highly dense,granular detergent base obtained such as by the spray-drying-granulationmethod, various ingredients may also be added after the preparation ofthe base.

When the detergent base is a liquid, it may be either a homogeneoussolution or an inhomogeneous dispersion. For removing the decompositionof carboxymethylcellulose by the cellulase in the detergent, it isdesirable that carboxymethylcellulose is granulated or coated before theincorporation in the composition.

The detergent compositions of this invention are used in industrial andhousehold uses at temperatures and liquor ratios conventionally employedin these environments.

In addition to their use in laundry detergents, substantially pure EGIII cellulase described herein can additionally be used in a pre-washingstep in the appropriate solution at an intermediate pH where sufficientactivity exists to provide desired improvements in colorretention/restoration, softening and feel. When the substantially pureEG III cellulase is employed in a pre-soak (e.g., pre-wash) composition,either as a liquid, spray, gel or paste composition, the substantiallypure EG III cellulase is generally employed from about 0.01 to about 20weight percent based on the total weight of the pre-soak composition. Insuch compositions, a surfactant may optionally be employed and whenemployed, is generally present at a concentration of from about 0.01 toabout 20 weight percent based on the total weight of the pre-soak. Theremainder of the composition comprises conventional components used inthe pre-soak, i.e., diluent, buffers, other enzymes (proteases), and thelike at their conventional concentrations. Accordingly, such pre-soakcompositions comprise from about 0 to about 20 weight percent of asurfactant and from about 0.01 to about 20 weight percent ofsubstantially pure EG III cellulase.

Also, it is contemplated that the substantially pure. EG III cellulosedescribed herein can be used in home use as a stand alone compositionsuitable for restoring color to faded fabrics (see, for example, U.S.Pat. No. 4,738,682, which is incorporated herein by reference in itsentirety) as well as used in a spot-remover.

Additionally, it is further contemplated that the high activity underneutral to alkaline conditions for EG III cellulose would be beneficialin textile processes for treating cotton-containing fabrics (see U.S.Ser. Nos. 07/677,385 and 07/678,865 which are incorporated herein byreference in their entirety) as well as in silage and/or compostingprocesses.

The following examples are offered to illustrate the present inventionand should not be construed in any way as limiting the scope of thisinvention.

EXAMPLES

Example 1 demonstrates the isolation of the components other than EG IIIfrom Cytolase 123 Cellulase (a complete fungal cellulose compositionobtained from Trichoderma longibrachiatum and available from GenencorInternational, Inc., South San Francisco, Calif.) via purificationprocedures.

Example 1 Purification of Cytolase 123 Cellulase into CellulaseComponents

CYTOLASE 123 cellulase was fractionated in the following manner. Thenormal distribution of cellulase components in this cellulase system isas follows:

    ______________________________________                                        CBH I            45-55 weight percent                                         CBH II           13-15 weight percent                                         EG I             11-13 weight percent                                         EG II            8-10 weight percent                                          EG III           1-4 weight percent                                           BG               0.5-1 weight percent.                                        ______________________________________                                    

The fractionation was done using columns containing the followingresins: Sephadex G-25 gel filtration resin from Sigma Chemical Company(St. Louis, Mo.), QA Trisacryl M anion exchange resin and SP Trisacryl Mcation exchange resin from IBF Biotechnics (Savage, Md.). CYTOLASE 123cellulase, 0.5 g, was desalted using a column of 3 liters of SephadexG-25 gel filtration resin with 10 mM sodium phosphate buffer at pH 6.8.The desalted solution, was then loaded onto a column of 20 ml of QATrisacryl M anion exchange resin. The fraction bound on this columncontained CBH I and EG I. These components were separated by gradientelution using an aqueous gradient containing from 0 to about 500 mMsodium chloride. The fraction not bound on this column contained CBH IIand EG II. These fractions were desalted using a column of Sephadex G-25gel filtration resin equilibrated with 10 mM sodium citrate, pH 3.3.This solution, 200 ml, was then loaded onto a column of 20 ml of SPTrisacryl M cation exchange resin. CBH II and EG II were elutedseparately using an aqueous gradient containing from 0 to about 200 mMsodium chloride.

Example 2 Purification of EG III from Cytolase 123 Cellulase

Example 1 above demonstrated the isolation of several components fromCytolase 123 Cellulase. However, because EG III is present in very smallquantities in Cytolase 123 Cellulase, the following procedures wereemployed to isolate this component.

A. Large Scale Extraction of EG III Cellulase Enzyme

One hundred liters of cell free cellulase filtrate were heated to about30° C. The heated material was made about 4% wt/vol PEG 8000(polyethylene glycol, MW of about 8000) and about 10% wt/vol anhydroussodium sulfate. The mixture formed a two phase liquid mixture. Thephases were separated using an SA-1 disk stack centrifuge. The phaseswere analyzed using silver staining isoelectric focusing gels.Fractionation and enrichment were obtained for EG III and xylanase. Therecovered composition contained about 20 to 50 weight percent of EG III.

Regarding the above procedure, use of a polyethylene glycol having amolecular weight substantially less than about 8000 gave inadequateseparation; whereas, use of polyethylene glycol having a molecularweight substantially greater than about 8000 resulted in the exclusionof desired enzymes in the recovered composition. With regard to theamount of sodium sulfate, sodium sulfate levels substantially greaterthan about 10% wt/vol caused precipitation problems; whereas, sodiumsulfate levels substantially less than about 10% wt/vol gave poorseparation or the solution remained in a single phase.

B. Purification of EG III Via Fractionation

The purification of EG III is conducted by fractionation from a completefungal cellulase composition (CYTOLASE 123 cellulose, commerciallyavailable from Genencor International, South San Francisco, Calif.)which is produced by wild type Trichoderma longibrachiatum.Specifically, the fractionation is done using columns containing thefollowing resins: Sephadex G-25 gel filtration resin from, SigmaChemical Company (St. Louis, Mo.), QA Trisacryl M anion exchange resinand SP Trisacryl M cation exchange resin from IBF Biotechnics (Savage,Md.). CYTOLASE 123 cellulose, 0.5 g, is desalted using a column of 3liters of Sephadex G-25 gel filtration resin with 10 mM sodium phosphatebuffer at pH 6.8. The desalted solution, is then loaded onto a column of20 ml of QA Trisacryl M anion exchange resin. The fraction bound on thiscolumn contained CBH I and EG I. The fraction not bound on this columncontains CBH II, EG II and EG III. These fractions are desalted using acolumn of Sephadex G-25 gel filtration resin equilibrated with 10 mMsodium citrate, pH 4.5. This solution, 200 ml, is then loaded onto acolumn of 20 ml of SP Trisacryl M cation exchange resin. The EG III waseluted with 100 mL of an aqueous solution of 200 mM sodium chloride.

In order to enhance the efficiency of the isolation of EG III, it may bedesirable to employ Trichoderma longibrachiatum genetically modified soas to overexpress EG III and/or to be incapable of producing one or moreof EG I, EG II, CBH I and/or CBH II components. This will necessarilylead to more efficient isolation of EG III by, for example,fractionation and/or PEG extraction as described above. Production ofsome of these strains of Trichoderma longibrachiatum are disclosed inU.S. Ser. No. 07/668,640, filed Mar. 13, 1991. Examples of production ofsome of these strains of Trichoderma longibrachiatum as set forth inthat application are as follows:

Example 3 Selection for pyr4 mutants of Trichoderma longibrachiatum

The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, anenzyme required for the biosynthesis of uridine. The toxic inhibitor5-fluoroorotic acid (FOA) is incorporated into uridine by wild-typecells and thus poisons the cells. However, cells defective in the pyr4gene are resistant to this inhibitor but require uridine for growth. Itis, therefore, possible to select for pyr4 mutant strains using FOA. Inpractice, spores of Trichoderma longibrachiatum strain RL-P37(Sheir-Neiss G. and Montenecourt, B. S., 1984, Appl. Microbiol.Biotechnol. 20:46-53) were spread on the surface of a solidified mediumcontaining 2 mg/ml uridine and 1.2 mg/ml FOA. Spontaneous FOA-resistantcolonies appeared within three to four days and it was possible tosubsequently identify those FOA-resistant mutants which required uridinefor growth. In order to identify those mutants which specifically had adefective pyr4 gene, protoplasts were generated and transformed with aplasmid containing a wild-type pyr4 gene (see Examples 5 and 6).Following transformation, protoplasts were plated on medium lackinguridine. Subsequent growth of transformed colonies demonstratedcomplementation of a defective pyr4 gene by the plasmid-borne pyr4 gene.In this way strain GC69 was identified as a pyr4 mutant of strainRL-P37.

Example 4 Preparation of CBHI Deletion Vector

A cbh1 gene encoding the CBHI protein was cloned from genomic DNA usingstrain RL-P37 by hybridization with an oligonucleotide probe designed onthe basis of the published sequence for this gene using known probesynthesis methods (Shoemaker et al., "Molecular Cloning ofExo-cellobiohydrolase I Derived from Trichoderma longibrachiatum StrainL27", Bio/Technology 1, p. 691 (1983)). The cbh1 gene resides on a 6.5kb PstI fragment and was inserted into PstI cut pUC4K (purchased fromPharmacia Inc., Piscataway, N.J.) replacing the Kan^(r) gene of thisvector. The resulting plasmid, pUC4K::cbhl was then cut with HindIII andthe larger fragment of about 6 kb was isolated and religated to givepUC4K::cbhlΔH/H. This procedure removes the entire cbh1 coding sequenceand approximately 1.2 kb upstream and 1.5 kb downstream of flanking DNAfrom either side of the original PstI fragment.

The Trichoderma longibrachiatum pyr4 gene was cloned as a 6.5 kbfragment of genomic DNA in pUC18 following the methods of Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2^(nd) ED., ColdSprings Harbor Press. The plasmid pUC4K::cbhIΔH/H was cut with HindIIIand the ends were desphosphorylated with calf intestinal alkalinephosphatase. This end dephosphorylated DNA was ligated with the 6.5 kbHindIII fragment containing the Trichoderma longibrachiatum pyr4 gene togive pΔCBHIpyr4. See FIG. 4.

Example 5 Isolation of Protoplasts

Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract,2% glucose) in a 500 ml flask with about 5×10⁷ Trichodermalongibrachiatum GC69 spores (the pyr4mutant strain). The flask was thenincubated at 37° C. with shaking for about 16 hours. The mycelium washarvested by centrifugation at 2,750×g. The harvested mycelium wasfurther washed in 1.2M sorbitol solution and resuspended in 40 ml ofNovozym^(R) 234 solution (which is the tradename for a multicomponentenzyme system containing 1,3-alpha-glucanase, 1,3-beta-glucanase,laminarinase, xylanase, chitinase and protease from Novo Biolabs,Danbury Conn.) containing 5 mg/ml Novozym^(R) 234; 5 mg/ml MgSO₄.7H₂ O;0.5 mg/ml bovine serum albumin; 1.2M sorbitol. The protoplasts wereremoved from cellular debris by filtration through Miracloth(Calbiochem. Corp) and collected by centrifugation at 2,000×g. Theprotoplasts were washed three times in 1.2M sorbitol and once in 1.2Msorbitol, 50 mM CaCl₂, centrifuged and resuspended. The protoplasts werefinally resuspended at a density of 2×10⁸ protoplasts per ml of 1.2Msorbitol, 50 mM CaCl₂.

Example 6 Transformation of Fungal Protoplasts

200 μl of the protoplast suspension prepared in Example 5 was added to20 μl of EcoRI digested pΔCBHIpyr4 (prepared in Example 4) in TE buffer(10 mM Tris, pH 7.4; 1 mM EDTA) and 50 μl of a polyethylene glycol (PEG)solution containing 25% PEG 4000, 0.6M KCl and 50 mM CaCl₂. This mixturewas incubated on ice for 20 minutes. After this incubation period 2.0 mlof the above-identified PEG solution was added thereto, the solution wasfurther mixed and incubated at room temperature for 5 minutes. Afterthis second incubation, 4.0 ml of a solution containing 1.2M sorbitoland 50 mM CaCl₂ was added thereto and this solution was further mixed.The protoplast solution was then immediately added to molten aliquots ofVogel's Medium N (3 grams sodium citrate, 5 grams KH₂ PO₄, 2 grams NH₄NO₃, 0.2 grams MgSO₄.7H₂ O, 0.1 gram CaCl₂.2H₂ O, 5 μg α-biotin, 5 mgcitric acid, 5 mg ZnSO₄.7H₂ O, 1 mg Fe(NH₄)₂.6H₂ O, 0.25 mg CuSO₄.5H₂ O,50 μg MnSO₄.4H₂ O per liter) containing an additional 1% glucose, 1.2Msorbitol and 1% agarose. The protoplast/medium mixture was then pouredonto a solid medium containing the same Vogel's medium as stated above.No uridine was present in the medium and therefore only transformedcolonies were able to grow as a result of complementation of the pyr4mutation of strain GC69 by the wild type pyr4 gene present inpΔCBHIpyr4. These colonies were subsequently transferred and stabletransformants purified, on a solid Vogel's medium N containing as anadditive, 1% glucose.

Example 7 Analysis of the Transformants

DNA was isolated from the transformants obtained in Example 5 after theywere grown in the liquid Vogel's medium N containing 1% glucose. Thesetransformant DNA samples were further cut with a PstI restriction enzymeand subjected to agarose gel electrophoresis. The gel was then furtherblotted onto a Nytran membrane filter and hybridized with a ³² Plabelled pΔCBHIpyr4 probe. The probe was selected to identify the nativecbh1 gene as a 6.5 kb PstI fragment, the native pyr4 gene and any DNAsequences derived from the transforming DNA fragment. FIG. 5 outlinesdeletion of the Trichoderma longibrachiatum cbh1 gene by integration ofthe larger EcoR1 fragment from pΔCBHIpyr4 at the cbh1 locus on one ofthe Trichoderma longibrachiatum chromosomes.

The bands from the hybridization were visualized via autoradiography.The result of the autoradiograph is seen in FIG. 6. Five samples wererun as described above, hence samples A, B, C, D, and E. Lane E is theuntransformed strain GC69 and was used as a control in the presentanalysis. Lanes A-D represent transformants obtained from the methodsdescribed above. The numbers on the side of the autoradiograph representthe sizes of molecular weight markers. As can be seen from thisautoradiograph, Lane D does not contain the 6.5 kb CBHI band, indicatingthat this gene has been totally deleted in the transformant. This cbh1deleted strain is called P37PΔCBHI. The other transformants analyzedappear identical to the untransformed control strain. Presumably, thishappened because the linear fragment from pΔCBHIpyr4 integrated by adouble cross-over at the native pyr4 locus to give a gene replacementevent.

Example 8

The same procedure was used in this example as in Example 7, except thatthe probe used was changed to a ³² P labelled pIntCBHI probe. This probeis a pUC-type plasmid containing a 2 kb BglII fragment from the cbh1locus within the region that was deleted in pUC4::cbh1ΔH/H. Two sampleswere run in this example including a control sample A, which is theuntransformed strain GC69 and the transformant P37PΔCBHI, sample B. Ascan be seen in FIG. 7, sample A contained the cbh1 gene, as indicated bythe band at 6.5 kb; however the transformant, sample B does not containthis 6.5 kb band and therefore does not contain the cbh1 gene.

Example 9 Protein Secretion by Strain P37PΔCBHI

Spores from the produced P37PΔCBHI strain were inoculated into 50 ml ofa Trichoderma basal medium containing 1% glucose, 0.14% (NH₄)₂ SO₄, 0.2%KH₂ PO₄, 0.03% MgSO₄, 0.03% urea, 0.75% bactotryptone, 0.05% Tween 80,0.000016% CuSO₄.5H₂ O, 0.001% FeSO₄.7H₂ O, 0.000128% ZnSO₄.7H₂ O,0.0000054% Na₂ MoO₄.2H₂ O, 0.0000007% MnCl.4H₂ O). The medium wasincubated while shaking in a 250 ml flask at 37° C. for about 48 hours.The resulting mycelium was collected by filtering through Miracloth(Calbiochem Corp.) and washed two or three times with 17 mM potassiumphosphate. The mycelium was finally suspended in 17 mM potassiumphosphate with 1 mM sophorose and further incubated for 24 hours at 30°C. while shaking. The supernatant was then collected from these culturesand the mycelium was discarded. Samples of the culture supernatant wereanalyzed by isoelectric focusing using a Pharmacia Phastgel system andpH 3-9 precast gels according to the manufacturer's instructions. Thegel was stained with silver stain to visualize the protein bands. Theband corresponding to the cbh1 protein was absent from the samplederived from the strain P37PΔCBHI, as shown in FIG. 8. This isoelectricfocusing gel shows various proteins in different supernatant cultures ofTrichoderma longibrachiatum. Lane A is partially purified CBHI; Lane Bis the supernatant from an untransformed Trichoderma longibrachiatumculture; Lane C is the supernatant from a strain deleted for the cbh1gene produced according to the methods of the present invention. Theposition of various cellulase components are labelled. Since CBHIconstitutes about 50% of the total extra-cellular protein, it is themajor secreted protein and hence is the darkest band on the gel. Thisisoelectric focusing gel clearly shows depletion of the CBHI protein inthe strain deleted for cbh1.

Example 10 Preparation of pPΔCBHII

The cbh2 gene of T. longibrachiatum, encoding the CBHII protein, hasbeen cloned as a 4.1 kb EcoRI fragment of genomic DNA which is showndiagrammatically in FIG. 9A (Chen et al., 1987, Biotechnology,5:274-278). Using methods known in the art, a plasmid, pPΔCBHII (FIG.9B), has been constructed in which a 3.2 kb central region of this clonebetween a HindIII site (at 74 bp 3' of the CBHII translation initiationsite) and a ClaI site (at 265 bp 3' of the last codon of CBHII) has beenremoved and replaced by the Trichoderma longibrachiatum pyr4 gene.

Digestion of this plasmid with EcoRI will liberate a fragment having 0.7kb of flanking DNA from the cbh2 locus at one end, 1.7 kb of flankingDNA from the cbh2 locus at the other end and the Trichodermalongibrachiatum pyr4 gene in the middle.

Example 11 Generation of a pyr4 mutant of P37PΔCBHI

Spores of the transformant (P37PΔCBHI) which was deleted for the cbh1gene were spread onto medium containing FOA. A pyr4⁻ derivative of thistransformant was subsequently obtained using the methods of Example 3.This pyr4⁻ strain was designated P37PΔCBHIPyr⁻ 26.

Example 12 Deletion of cbh2 gene in a strain previously deleted for cbh1

Protoplasts of strain P37PΔCBHIPyr⁻ 26 were generated and transformedwith EcoRI digested pPΔCBHII according to the methods outlined inExamples 5 and 6.

Purified stable transformants were cultured in shake flasks as inExample 9 and the protein in the culture supernatants was examined byisoelectric focusing. One transformant (designated P37PΔΔCBH67) wasidentified which did not produce any CBHII protein. Lane D of FIG. 8shows the supernatant from a strain deleted for both the cbh1 and cbh2genes produced according to the methods of the present invention.

DNA was extracted from strain P37PΔΔCBH67, digested with EcoRI andAsp718, and subjected to agarose gel electrophoresis. The DNA from thisgel was blotted to a membrane filter and hybridized with 32P labelledpPΔCBHII (FIG. 10). Lane A of FIG. 10 shows the hybridization patternobserved for DNA from an untransformed Trichoderma longibrachiatumstrain. The 4.1 kb EcoRI fragment containing the wild-type cbh2 gene wasobserved. Lane B shows the hybridization pattern observed for strainP37PΔΔCBH67. The single 4.1 kb band has been eliminated and replaced bytwo bands of approximately 0.9 and 3.1 kb. This is the expected patternif a single copy of the EcoRI fragment from pPΔCBHII had integratedprecisely at the cbh2 locus.

The same DNA samples were also digested with EcoRI and Southern analysiswas performed as above. In this example, the probe was ³² P labelledpIntCBHII. This plasmid contains a portion of the cbh2 gene codingsequence from within that segment of cbh2 DNA which was deleted inplasmid pPΔCBHII. No hybridization was seen with DNA from strainP37PΔΔCBH67 showing that the cbh2 gene was deleted and that no sequencesderived from the pUC plasmid were present in this strain.

Example 13 Construction of pEGIpyr4

The Trichoderma longibrachiatum egl1 gene, which encodes EGI, has beencloned as a 4.2 kb HindIII fragment of genomic DNA from strain RL-P37 byhybridization with oligonucleotides synthesized according to thepublished sequence (Penttila et al., 1986, Gene 45:253-263). A 3.6 kbHindIII-BamHI fragment was taken from this clone and ligated with a 1.6kb HindIII-BamHI fragment containing the Trichoderma longibrachiatumpyr4 gene and a pUC-based plasmid cut with HindIII to give the plasmidpEGIyr4 (FIG. 11). Digestion of pEGIpyr4 with HindIII would liberate afragment of DNA containing only Trichoderma longibrachiatum genomic DNA(the egl1 and pyr4 genes) except for 24 bp of sequenced, synthetic DNAbetween the two genes and 6 bp of sequenced, synthetic DNA at one end(see FIG. 11).

Example 14 Transformants of Trichoderma longibrachiatum containingpEGIpyr4

A pyr4 defective mutant of Trichoderma longibrachiatum strain RutC30(Sheir-Neiss and Montenecourt, 1984, Appl. Microbiol. Biotechnol.20:46-53) was obtained by the method outlined in Example 3. Protoplastsof this strain were transformed with undigested pEGIpyr4 and stabletransformants were purified. Five of these transformants (designatedEP2, EP4, EP5, EP6, EP11), as well as untransformed RutC30 wereinoculated into 50 ml of YEG medium (yeast extract, 5 g/l; glucose, 20g/l) in 250 ml shake flasks and cultured with shaking for 2 days at 28°C. The resulting mycelium was washed with sterile water and added to 50ml of TSF medium (0.05M citrate-phosphate buffer, pH 5.0; Avicelmicrocrystalline cellulose, 10 g/l; KH₂ PO₄, 2.0 g/l; (NH₄)₂ SO₄, 1.4g/l; proteose peptone, 1.0 g/l; Urea, 0.3 g/l; MgSO₄.7H₂ O, 0.3 g/l;CaCl₂, 0.3 g/l; FeSO₄.7H₂ O, 5.0 mg/I; MnSO₄.H₂ O, 1.6 mg/l; ZnSO₄, 1.4mg/l; CoCl₂, 2.0 mg/l; 0.1 % Tween 80). These cultures were incubatedwith shaking for a further 4 days at 28° C. Samples of the supernatantwere taken from these cultures and assays designed to measure the totalamount of protein and of endoglucanase activity were performed asdescribed below.

The endoglucanase assay relied on the release of soluble, dyedoligosaccharides from Remazol Brilliant Blue--carboxymethylcellulose(RBB-CMC, obtained from MegaZyme, North Rocks, NSW, Australia). Thesubstrate was prepared by adding 2 g of dry RBB-CMC to 80 ml of justboiled deionized water with vigorous stirring. When cooled to roomtemperature, 5 ml of 2M sodium acetate buffer (pH 4.8) was added and thepH adjusted to 4.5. The volume was finally adjusted to 100 ml withdeionized water and sodium azide added to a final concentration of0.02%. Aliquots of Trichoderma longibrachiatum culture supernatant or0.1M sodium acetate as a blank (10-20 μl) were placed in tubes, 250 μlof substrate was added and the tubes were incubated for 30 minutes at37° C. The tubes were placed on ice for 10 minutes and 1 ml of coldprecipitant (3.3% sodium acetate, 0.4% zinc acetate, pH 5 with HCl, 76%ethanol) was then added. The tubes were vortexed and allowed to sit for5 minutes before centrifuging for 3 minutes at approximately 13,000×g.The optical density was measured spectrophotometrically at a wavelengthof 590-600 nm.

The protein assay used was the BCA (bicinchoninic acid) assay usingreagents obtained from Pierce, Rockford, Ill., USA. The standard wasbovine serum albumin (BSA). BCA reagent was made by mixing 1 part ofreagent B with 50 parts of reagent A. One ml of the BCA reagent wasmixed with 50 μl of appropriately diluted BSA or Trichodermalongibrachiatum culture supernatant. Incubation was for 30 minutes at37° C. and the optical density was finally measuredspectrophotometrically at a wavelength of 562 nm.

It is clear that some of the transformants produced increased amounts ofendoglucanase activity compared to untransformed strain RutC30. It isthought that the endoglucanases or exo-cellobiohydrolases produced byuntransformed Trichoderma longibrachiatum constitute approximately 20%and 70% respectively of the total amount of protein secreted. Thereforea transformant such as EP5, which produces approximately four-fold moreendoglucanase than strain RutC30, would be expected to secreteapproximately equal amounts of endoglucanase-type andexo-cellobiohydrolase-type proteins.

The transformants described in this example were obtained using intactpEGIpyr4 and will contain DNA sequences integrated in the genome whichwere derived from the pUC plasmid. Prior to transformation it would bepossible to digest pEGIpyr4 with HindIII and isolate the larger DNAfragment containing only Trichoderma longibrachiatum DNA. Transformationof Trichoderma longibrachiatum with this isolated fragment of DNA wouldallow isolation of transformants which overproduced EGI and contained noheterologous DNA sequences except for the two short pieces of syntheticDNA shown in FIG. 11. It would also be possible to use pEGIpyr4 totransform a strain which was deleted for either the cbh1 gene, or thecbh2 gene, or for both genes. In this way a strain could be constructedwhich would over-produce EGI and produce either a limited range of, orno, exo-cellobiohydrolases.

The methods of Example 14 could be used to produce Trichodermalongibrachiatum strains which would over-produce any of the otherendoglucanases normally produced by Trichoderma longibrachiatum (T.longibrachiatum).

Likewise, it may be desirable for the EG III is compositions describedabove to be further purified. For example, EG III protein isolated inprocedures A or B above can be further purified by utilizing materialobtained from procedure A in procedure B or vice versa. One particularmethod for further purifying EG III is by further fractionation of an EGIII sample obtained in part b) of this Example 2. The further fractionwas done on a FPLC system using a Mono-S-HR 5/5 column (available fromPharmacia LKB Biotechnology, Piscataway, N.J.). The FPLC system consistsof a liquid chromatography controller, 2 pumps, a dual path monitor, afraction collector and a chart recorder (all of which are available fromPharmacia LKB Biotechnology, Piscataway, N.J.). The fractionation wasconducted by desalting 5 ml of the EG III sample prepared in part b) ofthis Example 2 with a 20 ml Sephadex G-25 column which had beenpreviously equilibrated with 10 mM sodium citrate pH 4. The column wasthen eluted with 0-200 mM aqueous gradient of NaCl at a rate of 0.5ml/minute with samples collected in 1 ml fractions. EG III was recoveredin fractions 10 and 11 and was determined to be greater than 90% pure bygel electrophoresis. EG III of this purity is suitable for determiningthe N-terminal amino acid sequence by known techniques.

Substantially pure EG III has the following characteristics which arecompared to the other endoglucanases isolated from Trichodermalongibrachiatum.

                  TABLE I                                                         ______________________________________                                                 MW            pI    pH optimum.sup.1                                 ______________________________________                                        EG I       ˜47-49 kD 4.7 ˜5                                       EG II      ˜35 kD    5.5 ˜5                                       EG III     ˜25-28 kD 7.4 ˜5.5-6.0                                 ______________________________________                                         .sup.1. pH optimum determined by RBBCMC activity as per Example 3 below. 

As can be seen from the above table, EG III has both a higher pH optimumand a higher pI as compared to the other endoglucanase components ofTrichoderma longibrachiatum. In Example 3 below, it is seen that EG IIIalso retains significant RBB-CMC activity under alkaline pHs.

Likewise, EG III cellulase from other strains of Trichoderma spp. can bepurified. For example, EG III cellulase derived from Trichoderma viridehas been described by Voragen et al., Methods in Enzymology, 16:243-249.This reference describes the EG III cellulase as having a molecularweight of about 23.5 Kdaltons, a pH optimum of 5.5, and a pI of 7.7.

Example 15 Activity of Cellulase Compositions over a pH Range

The following procedure was employed to determine the pH profiles of twodifferent cellulase compositions. The first cellulase composition was aCBH I and II deleted cellulase composition prepared from Trichodermalongibrachiatum genetically modified in a manner similar to thatdescribed above so as to be unable to produce CBH I and CBH IIcomponents. Insofar as this cellulase composition does not contain CBH Iand CBH II which generally comprise from about 58 to 70 percent of acellulose composition derived from Trichoderma longibrachiatum, thiscellulose composition is necessarily enriched in EG components. Since EGIII is the most minor of the endoglucanase components of Trichodermalongibrachiatum, this composition predominates in EG I and EG IIcomponents.

The second cellulase composition was an approximately 20-40% purefraction of EG III isolated from a cellulase composition derived fromTrichoderma longibrachiatum via purification methods similar to part b)of Example 2.

The activity of these cellulase compositions was determined at 40° C.and the determinations were made using the following procedures.

Add 5 to 20 μl of an appropriate enzyme solution at a concentrationsufficient to provide the requisite amount of enzyme in the finalsolution. Add 250 μl of 2 weight percent RBB-CMC (Remazol Brilliant BlueR-Carboxymethyl-cellulose--commercially available from MegaZyme, 6Altona Place, North Rocks, N.S.W. 2151, Australia) in 0.05Mcitrate/phosphate buffer at pH 4, 5, 5.5, 6, 6.5, 7, 7.5 and 8.

Vortex and incubate at 40° C. for 30 minutes. Chill in an ice bath for 5to 10 minutes. Add 1000 μl of methyl cellosolve containing 0.3M sodiumacetate and 0.02M zinc acetate. Vortex and let sit for 5-10 minutes.Centrifuge and pour supernatant into cuvets.

Relative enzyme activity was determined by measuring the optical density(OD) of the solution in each cuvet at 590 nm. Higher levels of opticaldensity correspond to higher levels of enzyme activity.

The results of this analysis are set forth in FIG. 1 which illustratesthe relative activity of the CBH I and II deleted cellulase compositioncompared to the EG III cellulase composition. From this figure, it isseen that the cellulase composition deleted in CBH I and CBH IIpossesses optimum cellulolytic activity against RBB-CMC at near pH 5.5and has some activity at alkaline pHs, i.e., at pHs from above 7 to 8.on the other hand, the cellulose composition enriched in EG IIIpossesses optimum cellulolytic activity at about pH 5.5-6 and possessessignificant activity at alkaline pHs.

Example 16 Isoelectric Focusing Gels

The purpose of this example is to illustrate isoelectric focusing gelsof different EG III cellulase compositions. Specifically, cellulaseproduced by a wild type Trichoderma longibrachiatum; cellulase derivedfrom a strain of Trichoderma longibrachiatum transformed so as to beincapable of expressing EG I and EG II cellulase proteins; andsubstantially pure EG III cellulase were analyzed on isoelectricfocusing gels.

Samples of these cellulases were analyzed by isoelectric focusing usinga Pharmacia IEF system (FBE-3000, Pharmacia Inc., Piscataway, N.J.) andpH 3-10 precast gels (Servalyt Precote, available from Serva, Carl-Berg,Germany) according to the manufacturer's instructions. The gels werestained with Ephortec™ stain (Serva Blue W, available from Serva FineBiochemicals, Westbury, N.Y. 11590) stain to visualize the proteinbands. The resulting gel is set forth in FIG. 2 wherein Lane 1 of FIG. 2illustrates the isoelectric focusing gel of cellulase derived from awild strain Trichoderma longibrachiatum; Lane 2 illustrates theisoelectric focusing gel of cellulase derived from a strain ofTrichoderma longibrachiatum so as to be incapable of expressing EG I andII; and Lane 3 illustrates the isoelectric focusing gel of substantiallypure EG III cellulase. In this figure, the margin adjacent to Lane 1 ismarked to identify the bands corresponding to cellulose proteins so asto permit identification of the proteins.

From the above figure, it is seen that because of EG III's high pI, thisprotein is found in a region usually associated with other high pIcomponents such as high pI xylanases, high pI β-glucosidases, etc.Moreover, this figure demonstrates that EG III is not a degradationproduct of either EG I or EG II proteins because, in Lane 2 of thisfigure, these proteins are not present while the EG III protein is.

Example 17 Color Restoration

The ability of EG III cellulase to restore color in cotton-containingfabrics was analyzed in the following experiment. Specifically, reducedcolor clarity in a worn cotton fabric arises from the accumulation onthe fabric of surface fibers over a period of time. These fibers giverise to a faded and matted appearance for the fabric and accordingly,the removal of these fibers is a necessary prerequisite to restoring theoriginal sharp color to the fabric. In view of the above, thisexperiment determines the ability of EG III cellulase to restore colorby measuring the ability of the EG III cellulose to remove surfacefibers.

In this experiment, two different compositions were compared for theability to remove fiber. Specifically, the first composition containssubstantially pure EG III cellulase (prepared in a manner similar tothat set forth in Example 2) whereas the second composition contains noEG III cellulase or any other cellulase composition.

In this example, an appropriate amount of cellulase (if employed) wasadded to separate solutions of 400 ml of a 20 mM citrate/phosphatebuffer containing 0.5 ml of a non-ionic surfactant. Samples wereprepared and titrated so as to provide for samples at pH 6, pH 7, pH 8and pH 9. Each of the resulting solutions was then added to a separatelaunderometer canister. Into these canisters were added a quantity ofmarbles to facilitate fiber removal as well as a 7 inch×5 inch cottonfabric (100% woven cotton, available as Style No. 439W from TestFabrics, Inc., 200 Blackford Ave., Middlesex, N.J. 08846). The canisterwas then closed and the canister lowered into the launderometer bathwhich was maintained at 43° C. The canister was then rotated in the bathat a speed of at least about 40 revolutions per minute (rpms) for about1 hour. Afterwards, the cloth is removed, rinsed well and dried in astandard drier.

The so treated fabrics were then analyzed for fiber removal byevaluation in a panel test. In particular, the fabrics (unmarked) wererated for levels of fiber by 6 individuals. The fabrics were visuallyevaluated for surface fibers and rated on a 1 to 7 scale. The scale hasseven standards to allow for meaningful comparisons. The standards are:

    ______________________________________                                        Rating       Standard.sup.a                                                   ______________________________________                                        7            Fabric not treated with cellulase                                6            Fabric treated.sup.b with 8 ppm cellulase                        5            Fabric treated with 16 ppm cellulase                             4            Fabric treated with 20 ppm cellulase                             3            Fabric treated with 40 ppm cellulase                             2            Fabric treated with 50 ppm cellulase                             1            Fabric treated with 100 ppm cellulase                            ______________________________________                                         .sup.a In all of the standards, the fabric was a 100% cotton sheeting         standardized test fabric (Style No. 439W) available from Test Fabrics,        Inc., 200 Blackford Ave., Middlesex, NJ 08846                                 .sup.b For all samples treated with the same cellulase composition.           Cellulase concentrations are in total protein. The launderometer treatmen     conditions are the same as set forth in Example 16 above.                

The fabric to be rated was provided a rating which most closely matchedone of the standards. After complete analysis of the fabrics, the valuesassigned to each fabric by all of the individuals were added and anaverage value generated. In these results, lower numbers correspond toimproved fiber removal.

The results of this analysis are set forth in FIG. 3 which illustratesthat at acidic, neutral and alkaline pH's, substantially pure EG IIIcellulase provides for fiber removal.

What is claimed is:
 1. A detergent composition suitable for use withcotton-containing fabrics which comprises:a cleaning effective amount ofa surfactant or a mixture of surfactants suitable for preparing analkaline wash medium having a pH of from above 7 to about 10 and fromabout 0.01 to about 5 weight percent of a cellulase compositioncomprising at least 40 weight percent of endoglucanase III based on thetotal weight of exo-cellobiohydrolase, endoglucanase and β-glucosidaseproteins in the cellulase composition wherein said endoglucanase III isan endoglucanase component derived from any strain of Trichoderma spp.which produces endoglucanase III and having a pH optimum of about 5.5 to6.0, an isoelectric point of from about 7.2 to 8.0, and a molecularweight of about 23 to 28 Kdaltons.
 2. A detergent composition accordingto claim 1 wherein said cellulase composition comprises from about 0.05to about 2 weight percent of said detergent composition.
 3. A detergentcomposition according to claim 1 wherein said detergent composition isfree of exo-cellobio-hydrolase I component.
 4. A detergent compositionaccording to claim 3 wherein said cellulase composition is further freeof exo-cellobiohydrolase II component.
 5. A detergent compositionaccording to claim 1 wherein said cellulase composition is derived fromTrichoderma longibrachiatum.
 6. A detergent composition according toclaim 1 wherein said cellulase composition is derived from Trichodermaviride.
 7. A detergent composition according to claim 1 wherein saidsurfactant or mixture of surfactants is suitable for preparing analkaline wash medium having a pH of from about 7 to about 8.